小反刍兽疫病毒双抗原夹心时间分辨荧光免疫测定方法的建立
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摘要
通过优化大肠埃希菌密码子、优化表达条件等方法,在大肠埃希菌表达系统中成功获得了可溶性的N蛋白与NH融合蛋白。进一步基于纯化的可溶性N蛋白与NH融合蛋白建立了小反刍兽疫病毒双抗原夹心时间分辨荧光免疫测定方法。该方法敏感性高,特异性强,能够特异性的检测羊血清中的小反刍兽疫病毒抗体,与其他相关疫病病原无交叉反应;重复性试验组内与组间变异系数均小于10%。对292份临床血清样品进行检测,与法国IDVET竞争ELISA试剂盒比较,阳性样品符合率为92.47%,阴性样品符合率为97.26%,总的符合率为94.86%。该方法与传统的ELISA方法相比,具有敏感性、特异性相当,操作简单、快速,具有较高的推广应用价值。
To establish the antigen sandwich time-resolved fluoroimmunoassay method for detection of peste des petits ruminants virus antibody in sheep serum,this research obtained the soluble N protein and NH fusion protein in Escherichia coli,through optimization of codon and protein expression conditions.Further based on purified N activity protein and NH fusion protein,a antigen sandwich time-resolved fluoroimmunoassay method for detection of peste des petits ruminants virus antibody was established.This method can detect the antibodies of peste des petits ruminants virus in sheep serum quickly and quantitatively.It has high sensitivity and specificity,and has no cross reaction to other related sheep pathogens.The intrabatch and interbatch coefficients of variation are all below 10%.A total of 292 clinical serum samples were tested,and the competition ELISA kit(IDVET)was used for comparison.The coincidence rate of the positive samples was 92.47%,the coincidence rate of the negative samples was 97.26%,and the total coincidence rate was 94.86%.This method is simple,rapid,sensitive,specific and has high practical value and popularization value.
To establish the antigen sandwich time-resolved fluoroimmunoassay method for detection of peste des petits ruminants virus antibody in sheep serum,this research obtained the soluble N protein and NH fusion protein in Escherichia coli,through optimization of codon and protein expression conditions.Further based on purified N activity protein and NH fusion protein,a antigen sandwich time-resolved fluoroimmunoassay method for detection of peste des petits ruminants virus antibody was established.This method can detect the antibodies of peste des petits ruminants virus in sheep serum quickly and quantitatively.It has high sensitivity and specificity,and has no cross reaction to other related sheep pathogens.The intrabatch and interbatch coefficients of variation are all below 10%.A total of 292 clinical serum samples were tested,and the competition ELISA kit(IDVET)was used for comparison.The coincidence rate of the positive samples was 92.47%,the coincidence rate of the negative samples was 97.26%,and the total coincidence rate was 94.86%.This method is simple,rapid,sensitive,specific and has high practical value and popularization value.
引文
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[12]郭昌明,张群,刘亚静,等.双抗体夹心ELISA检测小反刍兽疫病毒抗原方法的建立及病原流行病学调查[J].中国兽医学报,2017,37(5):799-803.
[13]Wu X,Liu F,Li L.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Virus Genes,2016,52(3):422-427
[14]Li L,Wu X,Liu F.Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR[J].J Virol Methods,2016,148(1):131-133.
[15]Holzer B,Hodgson S,Logan N.Protection of cattle against rinderpest by vaccination with wild-type but not attenuated strains of peste des petits ruminants virus.[J].J Virol,2016,90(10):5152-5162.
[2]李林杰,谢军,徐文凯,等.小反刍兽疫病毒的研究进展[J].黑龙江畜牧兽医,2018(1):66-70.
[3]赵柏林,孔冬妮,孙雨,等.我国小反刍兽疫流行情况及防控措施[J].畜牧与兽医2017,49(3):108-110.
[4]孙雨,赵柏林,王晓英,等.小反刍兽疫病毒N蛋白原核表达与间接ELISA检测方法的建立[J].动物医学进展,2017,39(1):6-10.
[5]Boussini H,Chitsungo E,Bodjo S C.First report and characterization of peste des petits ruminants virus in Liberia,West Africa[J].Trop Anim Health Prod,2016,48(7):1503-1507.
[6]Rafael G M,James J M,Alice B.RNA-binding specificity landscapes of designer pentatricopeptide repeat proteins elucidate principles of PPR-RNA interactions[J].Nucleic Acids Res,2018,46(5):2613-2623.
[7]Khic H P,Rachel C,Marie B,et al.The use of public performance reporting by general practitioners:a study of perceptions and referral behaviours[J].BMC Fam Pract,2018,19(29):1-11.
[8]Nuria A C,Conceptualization,Data C,et al.Optimization of non-denaturing protein extraction conditions for plant PPR proteins[J].PLoS One,2017,12(11):1877.
[9]Yirong W,Jianhua Y,Qingzhen Z,et al.The Schizosaccharomyces pombe PPR protein Ppr10associates with a novel protein Mpa1and acts as a mitochondrial translational activator[J].Nucleic Acids Res,2017,45(6):3323-3340.
[10]王净,王鹏,王长顺,等.小反刍兽疫病毒全基因组遗传进化分析[J].基因组学与应用生物学,2018(4):1393-1396.
[11]Damien G,Mauricio L O,Kevin B,et al.Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria[J].Proc Natl Acad,2017,114(33):8877-8882.
[12]郭昌明,张群,刘亚静,等.双抗体夹心ELISA检测小反刍兽疫病毒抗原方法的建立及病原流行病学调查[J].中国兽医学报,2017,37(5):799-803.
[13]Wu X,Liu F,Li L.Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed indirect ELISA:Practical considerations[J].Virus Genes,2016,52(3):422-427
[14]Li L,Wu X,Liu F.Rapid detection of lineage IV peste des petits ruminants virus by real-time RT-PCR[J].J Virol Methods,2016,148(1):131-133.
[15]Holzer B,Hodgson S,Logan N.Protection of cattle against rinderpest by vaccination with wild-type but not attenuated strains of peste des petits ruminants virus.[J].J Virol,2016,90(10):5152-5162.